title: Flow cytometry-based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology.
abstract: The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Sycuro
authors.forename: Chelsea S
authors.initials: CS
authors.lastname: Rule
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Petersen
authors.forename: Timna J
authors.initials: TJ
authors.lastname: Wyckoff
authors.forename: Tate
authors.initials: T
authors.lastname: Sessler
authors.forename: Dilip B
authors.initials: DB
authors.lastname: Nagarkar
authors.forename: Fakhra
authors.initials: F
authors.lastname: Khalid
authors.forename: Zachary
authors.initials: Z
authors.lastname: Pincus
authors.forename: Jacoby
authors.initials: J
authors.lastname: Biboy
authors.forename: Waldemar
authors.initials: W
authors.lastname: Vollmer
authors.forename: Nina R
authors.initials: NR
authors.lastname: Salama
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005075:Biological Evolution; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement; D002473:Cell Wall / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005798:Genes, Bacterial; D016480:Helicobacter pylori / Q000166:cytology* / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D010457:Peptidoglycan / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; C051467:peptidoglycan endopeptidase; D002268:Carboxypeptidases; D010450:Endopeptidases